User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/10

Cellular Adhesion

• Liquid Culture
  • IgAbc #8 did not grow up correctly
  • New tube with 8ml Amp/Cl LB, inoculated from 8.9 colony PCR backup plate
  • Put in incubator at 8:30 AM

• Glycerol
  • Took 1.6ml of liquid culture and created 10% glycerol stocks
    • GCN4 #1
    • GCN4 #3

• Miniprep
  • Extracted DNA from remaining liquid culture (~6ml)
  • Eluted in 50ul water

• Sequencing
  • 8.10.2007
  • All sequences were incorrect

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
    • C + D with B with IgAb-F and IgAb-R
    • B + C with IgAb-F and Mut_Pst1b-R
    • C + D with Mut_Pst1a-F and IgAb-R
  • Same thermocycler protocol as 8.1.2007

• Digest
  • Beginning to assemble parts into a functional construct
  • Template: 200ng DNA, 2ul NEB2, .2ul BSA, .5ul of each enzyme, rest water (20ul)
  • The first part should be cut with EcoR1 and Spe1
  • The second part should be cut with Xba1 and Pst1
    • IgAss #3: 8ul miniprep DNA, .5ul EcoR1, .5ul Spe1
    • Fos #3: 8ul miniprep DNA, .5ul Xba1, .5ul Spe1
    • JunB #2: 6ul miniprep DNA, .5ul Xba1, .5ul Spe1
  • Protocol: 2hr@37C, 20mins@80C (only 1hr@37 necessary)

B+C, C+D, C+D with B, log-2 ladder

• Gel
  • Ran 1.5% for 30 minutes at 100V to see if ligations worked
  • I think that Parts II and III are getting lost during PCR purification

• Ligation
  • Template: .5ul 1AK3 vector, 1ul ligation buffer, .5ul ligase, 2ul of each digest product, rest water(10ul rxn)
    • IgAss #3 and Fos #3
    • IgAss #3 and JunB #2
  • 30mins@roomtemp,10mins@65C

• Transformation
  • 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
  • 45secs heat shock at 42C using water bath
  • Added 300ul of SOC
  • Incubated for 1hr at 37C
  • Plated on Kan(out of Amp/Kan plates) and grew up at 37C overnight

• Antibody tags
  • Investigate creating antibody tags to see if IgA is being properly expressed on the surface of the cell
    • 6His (HHHHHH)
    • FLAG (DYKDDDDK)
    • Myc (EQKLISEEDL)
    • HA (YPYDVPDYA)
    • GST (220 aa)
  • All have Anti- from mouse